The protective function of the skin is dependent on epidermal terminal differentiation, the transition from the living cells of the granular layer to the anucleate cells of the stratum corneum. Many molecular events occur rapidly and in a coordinated manner at this complex transition point. The overall goals of this project are to determine mechanisms responsible for terminal differentiation of epidermal cells, with emphasis on the use of the filaggrin pathway and the interaction of filaggrin and keratin filaments during this transition. Hypotheses to be tested include: that filaggrin significantly alters the cytoskeleton and is a direct or indirect initiator of apoptosis, as related to terminal differentiation; that the rapid nature of the transition suggests that a triggering event is required; that this triggering event is likely to be a calcium dependent process and may be related to the initial enzymatic processing of profilaggrin by a member of the PC/furin family of calcium- dependent endoproteases; that specific sequences in profilaggrin are required for activation of filaggrin; that the profilaggrin amino- terminus functions in coordinating or relating some of the numerous events of terminal differentiation; that genetic disorders may delay or alter this transition. Studies will examine the role of filaggrin, as well as normal and mutant profilaggrin domains, in apoptosis using transient transfection and a tetracycline inducible transfection system in keratinocytes and/or COS cells, and in transgenic mice in which expression has been directed to spinous and granular layers of the epidermis. PC/furin enzymes will be identified in the epidermis and their involvement in profilaggrin processing evaluated. The project builds on several significant findings from the present project period and seeks to answer fundamental questions about terminal differentiation in epidermis, the role of filaggrin in this process, and the role of the filaggrin pathway in triggering this transition in normal skin and in genetic disorders.